The bHLH-PAS coactivator family of Nuclear Coactivators (NCoA; also known as p160/Steroid Receptor Coactivators, SRC) contains three related members: NCoA-1, NCoA-2 (also known as GRIP1), and NCoA-3 (also known as p/CIP, RAC3, ACTR, AIB1 or TRAM-1). Although the highest degree of sequence conservation exists within their bHLH and PAS domains, the coactivator function of the NCoA family has been ascribed to their C-terminal activation domains. These are employed to interact with a variety of nuclear receptors, other transcription factors, and different coactivators through conserved Nuclear Receptor boxes (NR boxes) defined as LXXLL motifs (where L is a conserved leucine and X represents any amino acid) (Heery et al., 1997
; Xu and Li, 2003
). The redundant interaction of NCoA members with an array of DNA-bound factors serves to efficiently recruit general coactivators such as CBP/p300 and CARM1/PRMT1 that possess potent histone modification activity needed for transcriptional activation.
Dimerization of NCoA family members plays an important role in the temporal regulation of gene transactivation by estrogen and androgen receptors. These nuclear receptors exhibit a bimodal pattern of gene regulation with rapid activation of a subset of genes that lack a classical hormone responsive element (HRE), and delayed activation of HRE-containing genes that require prolonged ligand-dependent stimulation (Perissi and Rosenfeld, 2005
). NCoA proteins are recruited as monomers to early gene, non-HRE-containing promoters, while dimeric complexes of NCoA-1/NCoA-3 and NCoA-2/NCoA-3 are preferentially recruited to late gene promoters containing HREs (Zhang et al., 2004
). This suggests that the mode in which the nuclear receptor is recruited to DNA, either indirectly through interaction with a previously bound transcription factor or through direct binding to the HRE, creates different requirements for NCoA coactivator interactions.
Recruitment of dimeric NCoA complexes to HRE-bound nuclear receptors requires NCoA PAS-B domains (Zhang et al., 2004
); however, unlike their bHLH-PAS transcription factor counterparts, the NCoA PAS-B domains do not interact with one another (Lodrini et al., 2008
). Instead, NCoA heterodimerization is mediated by the intermolecular association of the PAS-B domain with conserved C-terminal NR boxes within activation domain 1 (AD1, also known as the CBP-interaction domain, CID) () (Lodrini et al., 2008
). The PAS-B domain of NCoA-1 also directly interacts with a NR box from the STAT6 (signal transducer and activator of transcription 6) transcription factor (Litterst and Pfitzner, 2001
; Litterst and Pfitzner, 2002
). Importantly, NCoA heterodimerization via the PAS-B/NR box intermolecular interaction effectively competes for interactions with both CBP/p300 and STAT6, demonstrating that the oligomeric status of NCoA proteins is likely capable of regulating the affinity for subsequent transcription factor and/or coactivator recruitment (Lodrini et al., 2008
Regulation of NCoA coactivators by PAS domain/helical interactions
The molecular basis for the NCoA PAS-B/NR box interaction has been elucidated with the co-crystal structure of the NCoA-1 PAS-B domain complexed with the STAT6 LXXLL peptide (Razeto et al., 2004
). Surprisingly, the interaction is mediated by the α-helical surface of the PAS-B domain, with the LXXLL motif bound within a hydrophobic cleft formed by a rearrangement of two short helices into a long, colinear helix (). The colinear arrangement of the Dα and Eα helices is unique to this system among all the PAS structures solved to date (Möglich et al., 2009
), suggesting that binding of the NR box may induce an allosteric conformational change within the canonical PAS domain fold. LXXLL motifs fold into amphipathic helices upon interaction (Shiau et al., 1998
), with the conserved leucines oriented on the same face of the helix to interact in a knobs-and-holes configuration with helices within the target protein, as seen in typical coiled coil interactions. Residues that flank the conserved LXXLL motifs also make energetically important contacts with the PAS-B domain (Seitz et al., 2008
) and thus are likely important for defining specificity within NCoA heterodimer pairs. As hetero- and homodimeric NCoA complexes may have differing capacities to recruit additional coactivators through their AD1/CID domains, it is likely that the interaction of NCoA PAS-B domains with LXXLL motifs has introduced a new level of complexity within the combinatorial code of transcriptional regulation by bHLH-PAS coactivators.